Overview of gene expression and gene regulation in eukaryotes

Question 1

what is the lac operon

Answer 1

a regulated prokaryotic promoter
- activated by CAP, repressed by lac repressor
- ideal conditions for expression = +lactose and -glucose

Question 2

euchromatin vs heterochromatin

Answer 2

euchromatin: opened chromatin, active genes found here
heterochromatin: closed chromatin, inactive genes found here, not accessible to transcriptional machinery

Question 3

what type of RNA does each RNA polymerase produce

Answer 3

RNA pol I: pre-rRNA
RNA pol II: mRNA, miRNA, snRNA
RNA pol III: tRNA, snRNA, 5S RNA U6, 7S RNA

Question 4

what is epigenetic regulation

Answer 4

chromatin-mediated regulation - to transcribe DNA chromatin must be opened

Question 5

what is the structure of eukaryotic polymerase II?

Answer 5

complexes of multiple polypeptides
- 12 polypeptides RBP1, RBP2 … RBP12
- RBP1 is the clamp domain which accommodates DNA and then is closed by a bridge
- contains a carboxy-terminal domain of CTD1 subunit
-

Question 6

What is the carboxyl-terminal domain (CTD)

Answer 6

• involved in regulatory interactions in initiation, release, elongation and processing of mRNA
• phosphorylation of the CTD allows RNA pol II to move along the DNA
• Ser residue in the CTD are phosphorylated upon transition from initiation to elongation
• not structures - long and wobbly

Question 7

What regulates transcribed genes?

Answer 7

• conserved basal promoter elements (core promoter sequences)
• promoter-proximal binding sites for activators
• distal enhancers and repressors
• chromatin structure

Question 8

What are the core promoter sequences in DNA?

Answer 8

1. TATA box - prevalent in highly transcribed genes
2. Initiators - poorly conserved
3. BRE or DPE - influence activity of promoter

Question 9

What components help RNA pol recognize promoters and correctly initiate transcription

Answer 9

• several GTFs assemble the PIC over core promoter sequences
• DNA helicase helps initiate transcription
• Protein kinase helps release the polymerase
• other factors help the polymerase elongate
• other factors move nucleosomes

Question 10

what are the general transcription factors of RNA pol II

Answer 10

TFIIA, TFIIB, TFIID, TFIIE, TFIIH

Question 11

What are the similarities between TATA and CpG island promoters

Answer 11

• transcription proceeds in one direction
• RNA pol II is loaded onto them by GTFs

Question 12

What are differences between TATA and CpG island promoters

Answer 12

TATA
- transcription initiates in only 1 direction
- more nucleosomes = harder to transcribe
CpG island
- transcription is initiated in both directions
- transcription initiates anywhere on the island
- less nucleosomes = easier to transcribe

Question 13

what is ChIP

Answer 13

chromatin immunoprecipitation
- multistep technique that determines which DNA the DNA binding proteins is attaching to
- with anti-RNA pol II antibodies shows the binding of RNA pol II to the DNA
- shows 2 peaks for CpG islands
- shows 1 peak for TATA

Question 14

How is the pre-initiation complex assembled?

Answer 14

• TFIID associates with the promoter followed by TFIIA and TFIIB
• the mediator facilitates joining RNA pol II to the GTFs
• RNA pol II attached to TFIIF is recruited, the non-phosphorylated CTD of RNA pol II establishes contact with several GTFs - now have the core PIC
• TFIIE and TFIIH are recruited - now have closed PIC
• assembly is now finished

Question 15

How does the PIC open and become the initially transcribing complex?

Answer 15

• TFIIH helicase activity opens the DNA double helix
• RNA pol II initiates transcription
• TFIIH kinase activity phosphorylates the CTD to release RNA pol II from the promoter
• elongation can now commence

Question 16

transition from transcription initiation to elongation

Answer 16

• after TFIIH phosphorylates CTD, 2 elongation factors NELF and DSIF associate with RNA pol II and pause it downstream of the initiation site
• at this point another kinase PTEFb (aka CDH9/CycT) phosphorylates the CTD and NELF to get rid of it
• once RNA pol is released elongation can begin

Question 17

What does massive parallel RNA sequencing show

Answer 17

• the intensity of transcription over analyzed DNA
• tells you if we have synthesis of RNA in both sense and anti-sense strand

Question 18

what are the 3 capabilities of TFIIH

Answer 18

• helicase activity
• kinase activity
• DNA repair - fix nucleotide mismatch

Question 19

how can defects in transcriptional elongation lead to HIV

Answer 19

• HIV supresses PTEFb activity by a virally encoded protein called TAT
• TAT releases PTEFb and the polymerase transcribes the virus

Question 20

How do NELF and DSIF pause only viral transcription? IMPORTANT

Answer 20

-NELF and DSIF pause the polymerase after initiation
- further phosphorylation of CTD by PTEFb releases the polymerase and elongation begins
- upon initiation at 5’ end viral DNA has TAR which host RNA doesn’t have
- TAR allows for the virally encoded protein TAT to associate with it and block the activity of PTEFb
- PTEFb can’t phosphorylate NELF or CTD so viral DNA transcription is held
- cell stress occurs and is sensed by TAT - TAT falls off
- normal transcription continues

Question 21

What are the protein-binding transcription control regions that regulate gene expression

Answer 21

promoters: direct binding of RNA pol to DNA, multiple elements close to initiation site
promoter-proximal elements: close to promoter, inhibit transcription of genes
enhancers: increase transcription, could be upstream or downstream of initiation site
activators: boost gene transcription
repressors: decrease gene transcription

Question 22

what are differences between promoters and enhancers

Answer 22

Promoters
- function within a short distance
- immediately upstream from the initiation site
- position-dependent: non-functional if moved
- orientation dependent: drive transcription in only one direction
Enhancers
- can function over a long distance
- can be upstream, downstream or within introns
- position independent: functional when moved
- orientation independent: function in normal or inverted orientation

Question 23

How do enhancers communicate with promoters?

Answer 23

by bending DNA